Preliminary Analysis Of The Type Vi Secretion System In Vibrio Fluvialis

In Gram-negative bacteria, macromolecular proteins, including virulence factors,are released out of the cells or into the targeting cells by passing two membranes.Thus Gram-negative bacteria accomplish this complex process with the help of secretion system. To date, six secretion systems have been identified. The type VI secretion system (T6SS) is a recently discovered mechnism in Gram-negative bacteria that targets secreted proteins directly into eukaryotic as well as prokaryotic recipient cells in a contact-dependent manner, like type Ⅲ and type Ⅳ secretion systems (T3SS and T4SS) . Approximately 25% of all sequenced Gram-negative bacterial species encompass at last one gene cluster encoding T6SS. The cluster is composed of 15-20 genes which are conserved in different bacteria. The core genes include vasa, vasK,vasF, vasH, hcp, vgrG, vipA and vipB, forming a structrurally bacterial phage-like apparatus. Hcp and VgrG are not only components of the apparatus, but also secreated out of cells. VasH is σ54-dependent activators and regulate T6SS.In our previous study, we have accomplished genomic sequencing and analysis of an clinical isolate of Vibrio fluvialis,VF85003. Bioinformatic analysis predicted two gene clusters encoding T6SS in VF85003. But there still lacks experimental evidences to support that the type Ⅵ secretion system in Vibrio fluvialis is functional. In this study, we will investigate the function of type VI secretion system in Vibrio fluvialis.In order to verify and determine the conservation of gene organizational structure and the prevalence of T6SS clusters, we used PCR to selectively detect genes of two T6SS clusters in the Vibrio fluvialis strains isolated from different times, locations and sources. The results show that the gene loci of vasH, vasL/vask, vasA/vipB, predicted in Scaffold 10.1,present in all strains, and the arrangement is consistent with VF85003.Thus this means that the cluster is widespread and conserved in Vibriofluvialis. In addition, inVF85003 we amplified two additional hcp-vgrG coupled fragments using the sequence-specific primers designed on the basis of hcp-vgrG gene sequences of Vibrio furniss, which were respectively named as hcpA-vgrGA and hcpB-vgrGB. Thus, plus the originally predicted one copy of hcp-vgrG coupled gene sequence, VF85003 has at least three gene sequences encoding Hcp and VgrG protein.We also examined the transcription level of genes encoding T6SS by using realtime qRT-PCR. We find that when OD600 reaches 2.0, the mRNA expression level of gene vgrGA,vgrGB,vasK, vasF,and vasH is highest,and the mRNA expression levels of hcpA、hcpB、hcp and vgrG have two peaks at OD600 0.8 and 2.0, suggesting that transcriptional expression of genes encoding T6SS may be regulated by changes in the cell density.To illuminate the expression levels of three Hcp proteins and the effect of cell density on that,a pBBR-lux reporter was used to monitor the promotor activity of three copies of hcp. We found in VF85003 strain, promoter activity of hcpA was the highest which may indicate hcpA plays an important role. We also determined that VasH,a bEBP (bacterial enhancer binding protein), positively regulates hcp transcription. In addition, hcp expression is regulated by qurome sensing system.VflSfunctions as a quorum sensing regulator in Vbrio fluvialis, like HapR in Vibrio cholerae. Our results indicate that VflS accelerates hcp expression and probably VflR,another predicted transcriptional activator of quorum sensing in Vibrio fluvialis.Whether T6SS in Vibrio fluvialis can work as secretion system is still unknown.We used western-blot assay to detect Hcp protein. When cell density reached to OD6002.0 or even higher, we could detect Hcp protein in cell pellets. In the culture supernatant, we got a weak signal band similar to the size of Hcp protein but this remains to be further confirmed.Next step we used a label free quantitative mass spectrometry technique to understand the secretion function of T6SS by compared supernatant proteins from varies cultivate condition. The data showed the increased expression of flagella associate genes such as flaA、flaC、flaD under high salinity condition. Unfortunately we could not detect any Hcp, VgrG or other homolog proteins in T6SS. This may indicates T6SS might not have a secretion function under the present experiment conditions we used. Its biological function and environmental stimulus which may activate T6SS in VF85003 remains to be further investigated in our future study.